Rate Of Ribonucleic Acid Synthesis Research Articles

Base modifications affecting RNA polymerase and reverse transcriptase fidelity.

Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications, in both naturally-occurring post-transcriptional modifications and artificial chemical modifications used to expand the functionality of RNA. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity. Here, we describe the fidelity of RNA synthesis and reverse transcription of modified ribonucleotides using an assay based on Pacific Biosciences Single Molecule Real-Time sequencing. Several modified bases, including methylated (m6A, m5C and m5U), hydroxymethylated (hm5U) and isomeric bases (pseudouridine), were examined. By comparing each modified base to the equivalent unmodified RNA base, we can determine how the modification affected cumulative RNA polymerase and reverse transcriptase fidelity. 5-hydroxymethyluridine and N6-methyladenosine both increased the combined error rate of T7 RNA polymerase and reverse transcriptases, while pseudouridine specifically increased the error rate of RNA synthesis by T7 RNA polymerase. In addition, we examined the frequency, mutational spectrum and sequence context of reverse transcription errors on DNA templates from an analysis of second strand DNA synthesis.

Brain RNA synthesis, long-term potentiation and depression at the perforant path-granule cell synapse in the guinea pig

The effects of long-term changes in synaptic efficacy at the perforant path-granule cell synapse on the de-novo synthesis of ribonucleic acid (RNA) were investigated in hippocampal and cortical areas M anaesthetized Guinea pig preparations. Two experiments were run with stimulating and recording microelectrodes aimed at the perforant bundle and dentate gyrus hilus on both sides. In Experiment 1, a low-frequency (LFS; 0.02 Hz, 3 h) or high-frequency stimulation (HFS; 400 Hz, 250 ms) was delivered to the left perforant bundle with the contralateral side as control. In Experiment 2, animals received LFS or HFS trains with implanted nonstimulated animals used as controls. The latency and amplitude of the field postsynaptic potentials (FPSP) and population spike (POPS) were monitored under baseline conditions and following stimulation over a 3 h period. In addition, two HFS groups were tested with few (HFS-F: every 15 min) or several test stimuli (HFS-S: every 3 min). In both experiments RNA synthesis was determined by measuring the amount of 3H-5,6-uridbhe incorporated into the RNA 3 h after bilateral intraventrfcular injection. In Exp. 1 the LFS group showed a higher synthesis of RNA then both HFS groups. The rate of RNA synthesis did not differ between the stimulated and nonstimulated aide. In Exp. 2 the HFS groups showed a decreased RNA synthesis. In the HFS-F group, it pertained to the dorsal dentate area, CA1, subioulum, cingulate and dorsal cortices bilaterally, and to the ventral dentate sea and CA3 on the nonstimulated side. In contrast, the HFS-S group showed decreased RNA synthesis at the dorsal dentate area and dorsal cortex on the stimulated side, and at CA1, subiculum, and cingulate cortex bilaterally. The decrease was stronger in the HFS-F than in the HFS-S group. Moreover, the subgroup with a low (0–80°h) and that with a high (61–240%) level of long-term potentiation of FPSP revealed lower and higher RNA synthesis, respectively, both in homosynaptic target areas, and in heterosynaptic sites. Further, correlative analyses between FPSP, POPS and RNA synthesis revealed a complex pattern, depending upon the type of stimulation and on the brain side. Finally, cross-correlation analyses revealed a high degree of coupling among brain sites in the stimulated groups, indicating distributed covariant changes in RNA synthesis across different brain sites. Thus, changes in synaptic efficacy covay with changes In RNA syntImsis, and presumably exert a modulatory role on gene expression.

The transcription of various types of ribonucleic acid by hepatocytes isolated from rats of various ages.

The synthesis of various types of ribonucleic acid (RNA) isolated from 6- to 3-month-old female Fischer F344 rats was compared. The rate of RNA synthesis by freshly prepared hepatocytes was determined by dividing the amount of [3H]orotic acid incorporated into RNA as uridine-5'-monophosphate by the specific activity of the uridine-5'-triphosphate pool. The rate of total RNA synthesis by hepatocytes from 19-month-old rats was 40% less than the rate for hepatocytes from 12-month-old rats. No significant difference in the rate of total RNA synthesis was observed between 19 and 30 months of age. The percentage of [3H]orotic acid incorporated into poly(A) + RNA by 30-month-old rats was approximately 50% less than that observed for hepatocytes isolated from 6-month-old rats. The percentage of [3H]orotic acid incorporated into poly(A)-RNA as ribosomal RNA (38S, 18S, and 5S RNAs) or transfer RNA was similar for 12- and 30-month-old rats. The rate of poly(A) + RNA synthesis by hepatocytes isolated from 30-month-old rats was 65% less than that observed for hepatocytes from 6-month-old rats. In contrast to total RNA synthesis, the rate of poly(A) + RNA synthesis for the 30-month-old rats was significantly less than the rate for 19-month-old rats.

Transcriptional and post-transcriptional control of ribosomal protein and ribonucleic acid polymerase genes.

A partial restriction of ribonucleic acid (RNA) polymerase activity has been used to dissociate the coordinate synthesis of ribosomal proteins and subunits of RNA polymerase and to identify transcriptional and post-transcriptional control signals which regulate the expression of these component genes. Within the beta operon [which has the genetic organization: promoter (p beta), rplJ (L10), r;lL (L7/L12), attenuator, rpoB (beta), rpoC (beta'), terminator], the restriction caused a disproportionate increase between proximal and distal gene transcriptions; the transcriptional intensities of the proximal ribosomal protein genes and the distal RNA polymerase genes were elevated about two- and fourfold, respectively. Transcription within the operon containing four ribosomal protein genes and the RNA polymerase alpha gene was also enhanced, whereas transcription within operons containing only ribosomal protein genes was virtually unaffected by the restriction. It was thus concluded that the mechanisms controlling transcription initiation or attenuation or both in operons containing RNA polymerase subunit genes are coupled to the global rate of RNA synthesis. By introducing the composite ColE1 plasmid pJC701 carrying the proximal portion of the L10 operon, including the beta subunit gene, it was possible to achieve a 10- and a 30-fold range in the transcriptional intensities of the genes specifying L10 and L7/L12 and beta, respectively. Under these conditions, the relative synthesis rates of L7/L12 and beta protein varied by less than 2-fold and by about 15-fold, respectively. These observations corroborate the existence of a post-transcriptional mechanism which severely restricts translation of excess L7/L12 and L10 ribosomal protein messenger RNA; this mechanism is probably important in maintaining the balanced synthesis of ribosome components under conditions in which their messenger RNA levels are dissociated. Furthermore, the observed reduction in the translation efficiency of beta subunit messenger RNA may be related to an inhibitory effect caused by accumulation of RNA polymerase assembly intermediates.

RNA synthesis as a measure of microbial growth in aquatic environments. II. Field applications

Rates of stable ribonucleic acid (RNA) synthesis have been calculated from time-series measurements of the uptake of 3H-adenine and labeling of the intracellular adenosine triphosphate (ATP) and stable RNA pools for microbial communities from a variety of freshwater and marine ecosystems. Total rates of microbial RNA synthesis ranged from 6.6 pmol to 3.8x105 pmol of adenine incorporated into RNA l−1 h−1. Most of the variation was attributable to differences in microbial biomass; if total rate data are normalized to living microbial carbon (based on ATP measurements), the variation in specific rates of RNA synthesis is decreased by a factor of 100 to 1 000 for microbial communities from diverse environments. Light/dark experiments indicated that the uptake and incorporation of 3H-adenine are uncoupled from photophosphorylation. The measurement of rates of stable RNA synthesis of microbial assemblages in nature can yield useful information concerning in situ growth rates and the response of communities to changes in environmental conditions such as nutrient additions.

RNA synthesis as a measure of microbial growth in aquatic environments. I. Evaluation, verification and optimization of methods

Measurement of rates of microbial (bacteria and unicellular algae) ribonucleic acid (RNA) synthesis in environmental samples by radioactive tracers requires several assumptions that we test and evaluate herein. After addition of 3H-adenine to water samples from eutrophic freshwater or pelagic and oceanic ecosystems, microorganisms assimilate it without (1) any luxury uptake or (2) any expansion of intracellular adenine nucleotide pools. The calculated rate of stable RNA synthesis tends to be overestimated during the initial time periods of labeling (i.e., less than 30 s for cultures of Serratia marinorubra, and less than 20 min for most environmental samples) due to kinetic compartmentalization of the nucleic acid precursor (ATP) pool. Time-series analyses are recommended because the effects of compartmentalization decrease with increasing incubation. The kinetics of 3H-adenine uptake and aspects of its subsequent assimilation, intermediary metabolism and breakdown are described in bacteria and natural assemblages of microorganisms. Mass-balance radioactivity inventories greatly facilitate interpretation of nucleic acid precursor assimilation. Finally, we demonstrate that independent measurements of precursor (ATP) specific radioactivity are required for any elucidation of rates of stable RNA synthesis (and thus microbial growth) in nature.

Pattern of ribonucleic acid synthesis during germination of Allomyces macrogynus mitospores

The relationship between ribonucleic acid (RNA) synthesis and germination of Allomyces macrogynus mitospores was investigated. It was determined that the synthesis of all classes of RNA was initiated during the first 10 min of germination, around the time of encystment. It is during this stage that the membrane of the nuclear cap structure begins to break down, dispersing the cell complement of ribosomes throughout the cytoplasm. After encystment, there was an increase in the rate of synthesis of the four stable RNA species (4S, 5S, 19S, and 27S) which leveled off as the germ tube emerged. Data suggested that messenger RNA was synthesized at an increasing rate during the course of germination. Studies of RNA precursor pool behavior and RNA synthesis in the presence and absence of actinomycin D indicated that no species of RNA, including messenger RNA, was synthesized in the presence of actinomycin D. Further, precursor pool measurement indicated that the apparent increase in the rate of RNA synthesis during germination was largely due to increased specific activity of the RNA precursor pool.

Balanced growth and morphogenesis ofHistoplasma capsulatumin a defined synthetic medium

We have characterized balanced growth of yeast and hyphal cells of Histoplasma capsulatum and cells during transition from yeast to hyphae in a newly developed synthetic medium, R3B3. Homogeneous populations of yeast at 37 degrees C and hyphae at 25 degrees C grew in this medium with generation times of 10h and 19h, respectively. The growth rates were exponential, as demonstrated by the kinetics of net increase in dry weight. Identical rates of net increase were observed for ribonucleic acid (RNA) and protein, indicating conditions which approached steady state growth. In addition, this defined medium facilitated incorporation of radioactive precursors into RNA and protein and thus will allow for future detailed studies of macromolecular synthesis. When the incubation temperature of growing yeast cells was switched from 37 degrees C to 25 degrees C, the growth rate decreased as indicated in the generation time (19 h). Although the kinetic rates of RNA and protein synthesis also decreased, these rates were slightly faster relative to that observed for the increase in dry weight. During the later stage(s) of the yeast to hyphae transition a marked increase and subsequent decrease was observed for both RNA and protein. After this period of time, the synthesis of RNA and protein proceeded at the steady state rates usually observed in hyphal cultures.

Measurement of Microbial Activity and Growth in the Ocean by Rates of Stable Ribonucleic Acid Synthesis

A relatively simple and extremely sensitive technique for measuring rates of stable ribonucleic acid (RNA) synthesis was devised and applied to bacterial cultures and seawater samples. The procedure is based upon the uptake and incorporation of exogenous radiolabeled adenine into cellular RNA. To calculate absolute rates of synthesis, measurements of the specific radioactivity of the intracellular adenosine 5'-triphosphate pools (precursor to RNA) and of the total amount of radioactivity incorporated into stable cellular RNA per unit time are required. Since the rate of RNA synthesis is positively correlated with growth rate, measurements of RNA synthesis should be extremely useful for estimating and comparing the productivities of microbial assemblages in nature. Adenosine 5'-triphosphate, adenylate energy charge, and rates of stable RNA synthesis have been measured at a station located in the Columbian Basin of the Caribbean Sea. A subsurface peak in RNA synthesis (and therefore growth) was located within the dissolved oxygen minimum zone (450 m), suggesting in situ microbiological utilization of dissolved molecular oxygen. Calculations of the specific rates of RNA synthesis (i.e., RNA synthesis per unit of biomass) revealed that the middepth maximum corresponded to the highest specific rate of growth (420 pmol of adenine incorporated into RNA.day) of all depths sampled, including the euphotic zone. The existence of an intermediate depth zone of active microbial growth may be an important site for nutrient regeneration and may serve as a source of reduced carbon for mesopelagic and deep sea environments.

Specific inhibition of outgrowth of Bacillus subtilis spores by novobiocin

Spores of a Bacillus subtilis mutant temperature sensitive in deoxyribonucleic acid (DNA) replication proceeded through outgrowth at the nonpermissive temperature to the same extent as the wild-type parent spores. In contrast, the DNA synthesis inhibitor novobiocin completely prevented spore outgrowth while displaying a marginal effect on logarithmic growth during one generation time. Inhibition of outgrowth by novobiocin occurred in the absence of DNA replication, as demonstrated in an experiment with spores of the temperature-sensitive DNA synthesis mutant at the restrictive temperature. Novobiocin inhibited the initial rate of ribonucleic acid synthesis to the same extent in germinated spores and in exponentially growing cells. A novobiocin-resistant mutant underwent normal outgrowth in the presence of novobiocin. Therefore, novobiocin inhibition was independent of its effect on chromosome replication per se.

Bacterial lipopolysaccharide induction of a mouse spleen factor stimulating ribonucleic acid polymerase II

When bacterial lipopolysaccharide, a B-cell mitogen, was injected intraperitoneally into mice, the rate of deoxyribonucleic acid synthesis and the number of antibody-secreting cells in the spleen increased simultaneously, reaching a maximum in 3 days. The rate of ribonucleic acid synthesis also increased during this period, and this was found to be due to activation of alpha-amanitin-sensitive transcription in lymphoid cells of the spleen. The factors stimulating ribonucleic acid polymerase II in the spleens of normal mice and those treated with lipopolysaccharide were compared, and an additional factor besides that present in normal spleens was found in the spleen of lipopolysaccharide-treated mice.

Characteristics of pulse-labeled ribonucleic acid in relation to medium pH and acetate concentration during meiosis of yeast

Cellular impermeability associated with sporulating cells of Saccharomyces cerevisiae is caused by a rapid increase in the medium pH. Three factors have been identified as being important in regulating the rise in medium pH: 1) the cell density, 2) the potassium acetate concentration of the sporulation medium, and 3) and initial pH below 6.0. Sporulation conditions were established for strain 4579 which resulted in optimum uptake of 3H-adenine at T7, a period when the cells would be normally impermeable. Pulse-labeled polysomal RNA was characterized at T4 in naturally permeable cells of strain SK-1 and impermeable cells which required manipulation of the medium pH to facilitate uptake. Transfer ribonucleic acid (RNA), poly A-containing RNA and ribosomal RNA were synthesized in both cultures during the 20 min pulse. Furthermore, the rate of ribosomal RNA synthesis and processing into functional ribosomes approached the rate reported for vegetative cells. Initial sporulation conditions which caused a prolonged delay in the rise in medium pH adversely affected the kinetics of appearance and number of ascospores. The affect was shown to be on meiotic events since a reduction of sporulation was always accompanied by a reduction in the amount of intragenic recombination.

Bacillus subtilis mutant temperature sensitive in the synthesis of ribonucleic acid.

A Bacillus subtilis temperature-sensitive mutant (PB1653) has been isolated in which the rate of ribonucleic acid (RNA) synthesis sharply decreases after shift to 45 degrees C. Both stable and unstable RNAs are affected by the mutation. The possibility that the block of transcription at high temperature could be due to a "stringent" effect, mediated by an increase in the concentration of "magic spot" nucleotides, has been ruled out. Treatment with chloramphenicol (or streptomycin) rapidly restores the rate of RNA synthesis at 45 degrees C. The synthesis of RNA in the mutant during the early phases of spore germination is not temperature sensitive. The phage-specific transcription during infection with SPP1 phage, at high temperature, is less affected than that of the bacterial chromosome. In vitro experiments indicate that, in the mutant at high temperature, RNA polymerase undergoes a change in template specificity. The rna-53 mutation has been located on the B. subtilis genetic map near the hisA locus.

Calculation of absolute rates of RNA synthesis, accumulation, and degradation in tobacco callus in vivo.

Uptake and incorporation data were used to calculate an absolute rate of ribonucleic acid (RNA) synthesis by assuming that incorporation of radioactive nucleosides into RNA was a first-order function of the nucleoside triphosphate cellular pool. Homogenized tissue was separated into an RNA and cellular pool fraction. Methods were developed to directly measure the specific radioactivity of specific nucleosides in each fraction. Exogenous [3H]uridine was taken up by the cells, converted to I[3H]uridine triphosphate ([3H]UTP) and [3H]cytidine triphosphate ([3H]CTP), and incorporated into RNA. All of the radioactivity in RNA was contained in either uridine or cytidine. The average rate of RNA synthesis (20 mg of RNA (g of RNA)-1h-1) was calculated from changes in the specific radioactivity of uridine or cytidine in RNA and UTP or CTP in the pool. Since ribosomal RNA does not turn over in exponentially growing tissue, its rate of synthesis was measured (14 mg g-1h-1) and compared with the rate of accumulation of RNA (12 mg g-1h-1). The similarity between ribosomal RNA synthesis and total RNA accumulation confirmed the assumption that these methods measured an absolute rate of RNA synthesis.

Synthesis of deoxyribonucleic acid, ribonucleic acid, and protein during sporulation of Clostridium perfringens

The kinetics of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis as well as protein breakdown during sporulation by Clostridium perfringens were determined. Maximum levels of DNA and net RNA synthesis occurred 3 and 2 h, respectively, after inoculation of sporulation medium. The rate of RNA synthesis decreased as sporulation progressed. Deoxyadenosine increased uptake of [14C]uracil and [14C]thymine but depressed the level of sporulation and the formation of heat-resistant spores when added at concentrations above 100 mug/ml. Unlike Bacillus species, net protein synthesis, which was sensitive to chloramphenicol inhibition, continued during sporulation. The rate of protein breakdown during vegetative growth was 1%/h. During sporulation this rate increased to 4.7%/h. When added to sporulation medium at 0 time chloramphenicol reduced protein breakdown to 1%/h. If added at 3 h the rate decreased to 2.1%/h. The role of proteases in this process is discussed.

Macromolecular syntheses during biosynthesis of prodigiosin by Serratia marcescens

Amino acids that were utilized as sole sources of carbon and nitrogen for growth of Serratia marcescens Nima resulted in biosynthesis of prodigiosin in non-proliferating bacteria. Addition of alanine, proline, or histidine to non-proliferating cells incubated at 27 C increased the rate of protein synthesis and also caused biosynthesis of prodigiosin. No increase in the rate of protein synthesis was observed upon the addition of amino acids that did not stimulate prodigiosin biosynthesis. Increased rates of synthesis of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA) (a small amount) also occurred after addition of amino acids that resulted in biosynthesis of prodigiosin. After incubation of 24 h, the total amount of protein in suspensions of bacteria to which alanine or proline was added increased 67 and 98%, respectively. Total amounts of DNA and of RNA also increased before synthesis of prodigiosin. The amounts of these macromolecules did not increase after addition of amino acids that did not induce biosynthesis of progidiosin. However, macromolecular synthesis was not related only to prodigiosin biosynthesis because the rates of DNA, RNA, and protein synthesis also increased in suspensions of bacteria incubated with proline at 39 C, at which temperature no prodigiosin was synthesized. The quantities of DNA, RNA, and protein synthesized were lower in non-proliferating cells than in growing cells. The data indicated that amino acids causing biosynthesis of prodigiosin in non-proliferating cells must be metabolized and serve as sources of carbon and of nitrogen for synthesis of macromolecules and intermediates. Prodigiosin was synthesized secondarily to these primary metabolic events.

Stimulation of uterine RNA synthesis in mice by the post-coital antifertility agent, ORF 3858.

Stimulation of uterine ribonucleic acid (RNA) synthesis in mice by t he postcoital antifertility agent ORF 3858 was investigated. .1 mcg of ORF 3858 or its parahydroxylated congener ORF 5531 was injected sc into female mice which weighed 25-30 gm and had been ovariectomized 14-18 days previously. The mice received 6 mcCi tritiated uridine via the lateral tail vein at various times after the antifertility agent. At autopsy the uteri were removed. RNA synthesis was measured by a modified method of Miller and Emmens. The baseline rate of tritiated uridine incorporation in uterine RNA was 40 dpm/mg wet weight/hour. ORF 5531 caused an immediate decrease in the rate of RNA synthesis to a 4.7-fold peak within 8 hours. ORF 3858 remained without change at background levels for several hours before it increased to a maximum rate 2.5 times the background rate after 16 hours. 32 hours postinjection the rate induced by both returned to levels slightly below the baseline rates. It is concluded that the postcoital antifertility action fo ORF 3858 lies in its activity as a proestrogen.

Inhibition of de novo fatty acid synthesis by the antibiotic cerulenin in Bacillus subtilis: effects on citrate-Mg2+ transport and synthesis of macromolecules.

Inhibition of de novo fatty acid biosynthesis by the antibiotic cerulenin in Bacillus subtilis stopped de novo synthesis of neutral lipids and phospholipids. The bacteria ceased growing but remained completely viable. Addition of 12-methyltetradecanoic acid and palmitic acid to the culture medium of cerulenin-treated cells restored growth of the bacteria, albeit at a reduced rate. Although the de novo synthesis of all lipid components of the membrane was blocked, citrate-Mg(2+) transport activity remained inducible, and induced cells did not lose this transport activity when treated with cerulenin. Shortly after the addition of cerulenin, the rate of ribonucleic acid synthesis dropped rapidly and was followed by a slower decrease in the rate of protein synthesis. The rate of deoxyribonucleic acid synthesis remained almost unaffected. The rapid decrease of ribonucleic acid synthesis in cerulenin-treated cells might be due to the inhibition of de novo fatty acid biosynthesis or it might be due to a secondary effect of cerulenin in B. subtilis cells.

Mutants of Escherichia coli defective in membrane phospholipid synthesis: macromolecular synthesis in an sn-glycerol 3-phosphate acyltransferase Km mutant.

sn-Glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli have been selected from a strain which cannot aerobically catabolize G3P. The auxotrophy resulted from loss of the biosynthetic G3P dehydrogenase (EC 1.1.1.8) or from a defective membranous G3P acyltransferase. The apparent K(m) of the acyltransferase for G3P was 11- to 14-fold higher (from about 90 mum to 1,000 to 1,250 mum) in membrane preparations from the mutants than those of the parent. All extracts prepared from revertants of the G3P dehydrogenase mutants showed G3P dehydrogenase activity, but most contained less than 10% of the wild-type level. Membrane preparations from revertants of the acyltransferase mutants had apparent K(m)'s for G3P similar to that of the parent. Strains have been derived in which the G3P requirement can be satisfied with glycerol in the presence of glucose, presumably because the glycerol kinase was desensitized to inhibition by fructose 1,6-diphosphate. Investigations on the growth and macromolecular synthesis in a G3P acyltransferase K(m) mutant revealed that upon glycerol deprivation, net phospholipid synthesis stopped immediately; growth continued for about one doubling; net ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein nearly doubled paralleling the growth curve; the rate of phospholipid synthesis assessed by labeling cells with (32)P-phosphate, (14)C-acetate, or (3)H-serine was reduced greater than 90%; the rates of RNA and DNA synthesis increased as the cells grew and then decreased as the cells stopped growing; the rate of protein synthesis showed no increase and declined more slowly than the rates of RNA and DNA synthesis when the cells stopped growing. The cells retained and gained in the capacity to synthesize phospholipids upon glycerol deprivation. These data indicate that net phospholipid synthesis is not required for continued macromolecular synthesis for about one doubling, and that the rates of these processes are not coupled during this time period.

Decay of Ribonucleic Acid Synthesis in Amino Acid-Starved Escherichia coli After Rifampin Treatment

The concentration of rifampin necessary to affect the initiation of ribonucleic acid (RNA) synthesis quickly in Escherichia coli strains K-12 and 15TAU was about 200 mug/ml, as determined by extrapolation of the effect of the drug on the induction of beta-galactosidase synthesis. A lag in the action of rifampin of about 10 s was confirmed. Rifampin was then used as a probe to compare RNA synthesis in growing and amino acid-starved E. coli. Restoring arginine to arginine-starved strain 15TAU immediately after rifampin inhibition did not detectably restore the rate of uracil uptake to that of uninhibited cells. The residual rate of RNA synthesis (corrected for acid-soluble triphosphate specific activities) after rifampin treatment of both growing and isoleucine-starved (valine-inhibited) cultures of strain K-12 showed similar decay kinetics. These findings support the notion that amino acid starvation blocks the initiation of some RNA transcription units, but do not rule out other possibilities.